The vasa vasorum are a network of microvasculature that originate primarily in the adventitia of large arteries (Heistad & Marcus (1979) Blood Vessels 16(5):225-238). Neovascularized second order vasa vasorum are associated with more advanced stages of human atherosclerosis (O'Brien, et al. (1994) Am. J. Pathol. 145(4):883-894; Virmani, et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25(10):2054-2061). The presence and extent of vasa vasorum correlate with atherosclerotic lesion size and lumen diameter in hypercholesterolemic animal models (Heistad & Marcus (1979) supra; Khurana, et al. (2004) Circulation 110(16):2436-2443; Langheinrich, et al. (2007) Atherosclerosis 191(1):73-81; Langheinrich, et al. (2006) Arterioscler. Thromb. Vasc. Biol. 26(2):347-352; Moulton, et al. (2003) Proc. Natl. Acad. Sci. USA 100(8):4736-4741). It has been demonstrated that anti-angiogenic proteins inhibit neovascularization of the vasa vasorum and associated plaque progression in genetically modified mouse models of atherosclerosis (Moulton, et al. (2003) supra; Drinane, et al. (2009) Circ. Res. 104(3):337-345; Moulton, et al. (1999) Circulation 99(13):1726-1732). Each of these inhibitors is a cleavage product of an extracellular matrix (ECM) protein (Mulligan-Kehoe, et al. (2002) J. Biol. Chem. 277(50):49077-49089; Mulligan-Kehoe, et al. (2001) J. Biol. Chem. 276(11):8588-8596; O'Reilly, et al. (1997) Cell 88(2):277-285; O'Reilly, et al. (1994) Cell 79(2):315-328).
The ECM and basement membrane (BM) provide a support scaffold that is essential for blood vessel stability. Adhesion of endothelial cells (EC) to the ECM enables them to undergo migration, proliferation and morphogenesis, which are all necessary for neovascularization (Rhodes & Simons (2007) J. Cell Mol. Med. 11(2):176-205). Degradation of the ECM/BM leads to vessel collapse/regression (Davis & Saunders (2006) J. Investig. Dermatol. Symp. Proc. 11(1):44-56; Davis & Senger (2005) Circ. Res. 97(11):1093-1107; Vernon &Sage (1995) Am. J. Pathol. 147(4):873-883). Proteases that degrade the ECM/BM play a key role in matrix remodeling during normal wound healing as well as in vascular diseases such as atherosclerosis (Garcia-Touchard, et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25(6):1119-1127).
Plasmin contributes to matrix remodeling through its own proteolytic activity and by activating numerous matrix metalloproteinases (MMPs) (Garcia-Touchard, et al. (2005) supra; Bobik & Tkachuk (2003) Curr. Hypertens. Rep. 5(6):466-472). Of those, MMP-1, -3, -9, -10 and -13 promote capillary network regression (Davis, et al. (2001) J. Cell Sci. 114(Pt 5):917-930; Saunders, et al. (2005) J. Cell Sci. 118(Pt 10):2325-2340). Plasmin also contributes to ECM remodeling by degrading fibrin (Vernon &Sage (1995) supra; Pepper (2001) Arterioscler. Thromb. Vasc. Biol. 21(7):1104-1117; Senger (1996) Am. J. Pathol. 149(1):1-7), an ECM protein that forms a supportive scaffold for angiogenic vessels (Dvorak, et al. (1992) Ann. NY Acad. Sci. 667:101-111; Nagy, et al. (1989) Biochim. Biophys. Acta 948(3):305-326. Fibrin, the major constituent of provisional matrix (Werb (1997) Cell 91(4):439-442), enables endothelial cells to adhere, spread and proliferate (Cheresh, et al. (1989) Cell 58(5):945-953; Suchiro, et al. (1997) J. Biol. Chem. 272(8):5360-5366). Fibrinogen extravasates from “leaky” angiogenic vessels, then accumulates in the matrix where it can be converted to fibrin by thrombin (Mosesson (2005) J. Thromb. Haemost. 3(8):1894-1904). Fibrin or accumulated fibrinogen can be broken down by plasmin to negatively regulate angiogenesis (Sahni & Francis (2003) J. Thromb. Haemost. 1(6):1271-1277). The breakdown products are released into the plasma. Plasmin also degrades nidogen (Mayer, et al. (1993) Eur. J. Biochem. 217(3):877-884) and perlecan (Whitelock, et al. (1996) J. Biol. Chem. 271(17):10079-10086), two of the four key components of the ECM/BM. Nidogen is a sulfated glycoprotein that connects two of the other key BM components, laminin and type IV collagen. Perlecan is a large heparan sulfate proteoglycan whose core protein binds many molecules to include nidogen, type IV collagen, laminin and angiogenic growth factors, FGF-2 and VEGF. Nidogen, perlecan, laminin and type IV collagen are important in blood vessel formation (Rhodes & Simons (2007) supra).
Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of plasmin production. It functions in this capacity by exposing a reactive center loop (RCL) that binds tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). This interaction prevents the plasminogen activators from converting plasminogen to plasmin (Dellas & Loskutoff (2005) Thromb. Haemost. 93(4):631-640; Seiffert, et al. (1990) Cell Differ. Dev. 32(3):287-292).
It has been demonstrated that an anti-angiogenic truncated plasminogen activator inhibitor-1(PAI-1) protein, rPAI-123 (Mulligan-Kehoe, et al. (2002) supra; Mulligan-Kehoe, et al. (2001) supra; Drinane, et al. (2006) J. Biol. Chem. 281(44):33336-33344), significantly inhibits angiogenesis in cultured endothelial cells and aortic rings from chick embryos by stimulating a high level of endothelial cell-specific apoptosis (Mulligan-Kehoe, et al. (2002) supra; Mulligan-Kehoe, et al. (2001) supra; Drinane, et al. (2006) supra). Additionally, rPAI-123 inhibits angiogenic vasa vasorum, reduces plaque area and plaque cholesterol in the descending aorta (DA) of hypercholesterolemic LDLR−/− ApoB48 deficient mice (Drinane, et al. (2009) supra). In this model, adventitial vasa vasorum in the saline-treated group have a lumen and form a defined vascular network, which is disordered, disrupted and appears to be collapsing in rPAI-123-treated mice.